NRF2 Screening Assays and Related Methods and Compositions

ABSTRACT

Provided are certain methods of screening, identifying, and evaluating neuroprotective compounds useful for treatment of neurological diseases, such as, e.g., multiple sclerosis (MS). The compounds described upregulate the cellular cytoprotective pathway regulated by Nrf2. Also provided are certain methods of utilizing such compounds in therapy for neurological disease, particularly, for slowing or reducing demyelination, axonal loss, or neuronal and oligodendrocyte death.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/767,014, filed Feb. 14, 2013, which is a continuation of U.S. patentapplication Ser. No. 13/372,426, filed Feb. 13, 2012, now U.S. Pat. No.8,399,514, issued Mar. 19, 2013, which is a continuation of U.S. patentapplication Ser. No. 12/526,296, abandoned, which is the national stageof International Patent Application No. PCT/US2008/001602, filed Feb. 7,2008, which claims the benefit of U.S. Provisional Patent Application60/888,921, filed Feb. 8, 2007. U.S. patent application Ser. No.13/767,014, filed Feb. 14, 2013, is incorporated by reference herein inits entirety.

Provided are certain compounds for treating neurological diseases,including demyelinating neurological diseases, such as, e.g., multiplesclerosis.

Multiple sclerosis (MS) is an autoimmune disease with the autoimmuneactivity directed against central nervous system (CNS) antigens. Thedisease is characterized by inflammation in parts of the CNS, leading tothe loss of the myelin sheathing around neuronal axons (demyelination),loss of axons, and the eventual death of neurons, oligodenrocytes andglial cells.

An estimated 2,500,000 people in the world suffer from MS. It is one ofthe most common diseases of the CNS in young adults. MS is a chronic,progressing, disabling disease, which generally strikes its victims sometime after adolescence, with diagnosis generally made between 20 and 40years of age, although onset may occur earlier. The disease is notdirectly hereditary, although genetic susceptibility plays a part in itsdevelopment. Relapsing-remitting MS presents in the form of recurrentattacks of focal or multifocal neurologic dysfunction. Attacks mayoccur, remit, and recur, seemingly randomly over many years. Remissionis often incomplete and as one attack follows another, a stepwisedownward progression ensues with increasing permanent neurologicaldeficit.

Although various immunotherapeutic drugs can provide relief in patientswith MS, none is capable of reversing disease progression, and some cancause serious adverse effects. Most current therapies for MS are aimedat the reduction of inflammation and suppression or modulation of theimmune system. As of 2006, the available treatments for MS reduceinflammation and the number of new episodes but not all have an effecton disease progression. A number of clinical trials have shown that thesuppression of inflammation in chronic MS rarely significantly limitsthe accumulation of disability through sustained disease progression,suggesting that neuronal damage and inflammation are independentpathologies. Promoting CNS remyelination as a repair mechanism andotherwise preventing axonal loss and neuronal death are some of theimportant goals for the treatment of MS. For a comprehensive review ofMS and its current therapies, see, e.g., McAlpine's Multiple Sclerosis,by Alastair Compston et al., 4th edition, Churchill LivingstoneElsevier, 2006.

“Phase 2 enzymes” serve as a protection mechanism in mammalian cellsagainst oxygen/nitrogen species (ROS/RNS), electrophiles andxenobiotics. These enzymes are not normally expressed at their maximallevels and, their expression can be induced by a variety of natural andsynthetic agents. Nuclear factor E2-related factor 2 (Nrf2) is atranscription factor responsible for the induction of a variety ofimportant antioxidant and detoxification enzymes that coordinate aprotective cellular response to metabolic and toxic stress.

ROS/RNS are most damaging in the brain and neuronal tissue, where theyattack post-mitotic (i.e., non-dividing) cells such as glial cells,oligodendocytes, and neurons, which are particularly sensitive to freeradicals. This process leads to neuronal damage. Oxidative stress hasbeen implicated in the pathogenesis of a variety of neurodegenerativediseases, including ALS, Alzheimer's disease (AD), and Parkinson'sdisease (PD). For review, see, e.g., van Muiswinkel et al., Curr. DrugTargets CNS—Neurol. Disord., 2005, 4:267-281. An anti-oxidative enzymeunder control of Nrf2, NQO1 (NAD(P)H dehydrogenase, quinone (1), wasrecently reported to be substantially upregulated in the brain tissuesof AD and PD subjects (Muiswinkel et al., Neurobiol. Aging, 2004, 25:1253). Similarly, increased expression of NQO1 was reported in the ALSsubjects' spinal cord (Muiswinkel et al., Curr. Drug Targets—CNS.Neurol. Disord., 2005, 4:267-281) and in active and chronic lesions inthe brains of patients suffering from MS (van Horssen et al., FreeRadical Biol. & Med., 2006, 41 311-311). These observations indicatethat the Nrf2 pathway may be activated in neurodegenerative andneuroinflammatory diseases as an endogenous protective mechanism.Indeed, most recently, it has been reported that induced activation ofNrf2-dependent genes by certain cyclopenanone-based compounds (NEPP)counters the toxic effects of metabolic inhibition and ROS/RNSproduction in the brain and protects neurons from death in vitro and invivo (see Satoh et al., PNAS, 2006, 103(3):768-773).

Additionally, many publications have reported neuroprotective effects ofcompounds in natural plant-derived compounds (“phytochemicals”),including α-tocopherol (vitamin E), lycopene (tomatoes), resveratrol(red grapes), sulforaphane (broccoli), EGCG (green tea), etc. Forreview, see Mattson and Cheng, Trends in Neurosci., 2006,29(11):632-639. Originally, the action of these compounds was attributedto their anti-oxidant properties. However, while most anti-oxidants areeffective only at high concentrations, at least some of these compoundsappear to exert neuroprotective effects at much lower doses. Emergingevidence suggests that these compounds may exert their neuroprotectiveeffects by activating cellular stress-response pathways, including theNrf2 pathway, resulting in the upregulation of neuroprotective genes.However, the exact mechanism of action of these compounds remains poorlyunderstood.

To date, more than 10 different chemical classes of inducers of Nrf2pathway have been identified including isothiocyanates and their thioladdition products, dithiocarbamates, as well as 1,2-dithiole-3-thiones,trivalent arsenic derivatives (e.g., phenyl arsenoxide), heavy metals,certain conjugated cyclic and acyclic polyenes (including porphyrins,chlorophyllins, and chlorophyll), and vicinal dimercaptans. Theseinducers have few structural similarities. They are mostlyelectrophiles, and all can react chemically with thiol groups byalkylation, oxidation, or reduction, suggesting that the intracellularsensor for inducers is likely to contain very highly reactive (cysteine)thiols. The inducers can modify thiol groups by a variety of mechanismsincluding: alkylation (Michael addition acceptors, isothiocyanates,quinones); oxidation (e.g., peroxides and hydroperoxides); and directreaction with thiol/disulfide linkages (e.g., vicinal dithiols such as1,2-dimercaptopropanol, lipoic acid). These diverse response mechanismsprovide plasticity for cellular responses to a variety of electrophilicand oxidant stressors.

Provided are methods that comprise at least one of the followingmethods:

-   -   1) methods of screening for at least one new candidate compound        for treating a neurological disease;    -   2) methods of evaluating neuroprotective properties of at least        one drug candidate for treating a neurological disease;    -   3) methods of comparing (e.g., for bioequivalence) at least two        pharmaceutical compositions which comprise fumaric acid        derivatives;    -   4) methods of treating a neurological disease by administering        to the subject in need thereof at least one compound that is        partially structurally similar to DMF or MMF; and    -   5) methods of treating a neurological disease by a combination        therapy that comprises administration of at least one first        compound that upregulates the Nrf2 pathway and at least one        second compound that does not upregulate the Nrf2 pathway.

In some embodiments, the neurological disease is a neurodegenerativedisease such as, for example, ALS, Parkinson's disease, Alzheimer'sdisease, and Huntington's disease. In some embodiments the neurologicaldisease is MS or another demyelinating neurological disease.

In some embodiments, the methods 1-3 further comprise:

-   -   a) contacting a cell with the test compound, and    -   b) determining whether the Nrf2 pathway is upregulated in the        cell.        In some embodiments, the methods may further comprise:    -   c) determining whether the test compound slows or prevents        demyelination, axonal loss, and/or neuronal death, and/or    -   d) selecting the test compound as a candidate for treating        neurodegeneration in a neurological disease if 1) the Nrf2        pathway is upregulated and 2) demyelination, axonal loss, and/or        neuronal death are/is prevented or slowed.

In some embodiments, the methods 1-3 comprise contacting a cell with atleast one test compound and determining whether the Nrf2 pathway isupregulated in the cell. In such methods, an upregulation of the Nrf2pathway above a threshold (e.g., by at least 30% over a control)indicates that the at least one compound has at least one biologicalproperty beneficial in treating a neurological disease (e.g.,neuroprotective properties). In some embodiments, the upregulation ofthe Nrf2 pathway is assessed (in vivo and/or in vitro) by at least oneof the following:

-   -   i) expression levels of endogenously produced and/or exogenously        introduced Nrf2;    -   ii) subcellular localization and/or nuclear translocation of        Nrf2;    -   iii) expression levels and/or activity of one or more genes        under control of Nrf2 (e.g., endogenous NQO1) or an        Nrf2-regulated reporter gene in an artificial reporter        construct;    -   iv) levels of Nrf2 binding to the Nrf2-binding DNA element ARE;    -   v) stability of Nrf2/Keap1 complexes; and    -   vi) modification (e.g., alkylation) levels of Keap1 and/or at        least one other Nrf2/Keap1-associated proteins.

In some embodiments of methods 1-3, the compounds that are beingscreened, evaluated, or compared comprise at least one member of atleast one of the following classes of compounds: mild alkylating agents,Michael addition acceptors, and compounds that are metabolized uponadministration to Michael addition acceptors. In some embodiments, theMichael addition acceptor has the structure of Formula I, II, III, or IVset forth below.

In some embodiments method 1 comprises:

-   -   a) contacting a cell with a plurality of test compounds,    -   b) determining whether the Nrf2 pathway is upregulated in the        cell, and    -   c) selecting from the plurality of compounds at least one        compound that upregulates the Nrf2 pathway,        wherein an upregulation of the Nrf2 pathway by the selected at        least one compound indicates that the selected at least one        compound may be useful for treating a neurological disease. The        plurality of compounds may be represented, e.g., by a        combinatorial chemical library, and the method may be performed,        e.g., by high-throughput screening.

In some embodiments method 2 comprises:

a) contacting a cell with the at least one drug or drug candidate, and

b) determining whether the Nrf2 pathway is upregulated in the cell,

wherein an upregulation of the Nrf2 pathway by the at least one drug ordrug candidate indicates that the at least one drug or drug candidate isuseful for neuroprotection in treating a human having a neurologicaldisease.

In some embodiments method 3 comprises:

-   -   a) contacting a cell with a first composition comprising at        least one test compound, and    -   b) comparing the level of Nrf2 pathway upregulation in the cell        by the at least one test compound to the corresponding level of        the Nrf2 pathway upregulation in a control cell treated with a        second composition comprising at least one of DMF and MMF.

In some embodiments of method 3, the test compound is fumaric acid, asalt thereof, or a fumaric acid derivative. In some embodiments, thefirst composition comprises DMF, MMF, or both. In some embodiments, thedose and/or the formulation of the first composition differs from thedose and/or the formulation of the second composition.

In some embodiments, method 3 further comprises:

c) comparing at least one pharmacokinetic parameter (e.g.,serum-half-life) of the first and the second compositions.

In some embodiments method 4 comprises administering to the mammal atherapeutically effective amount of at least one neuroprotectivecompound having Formula I, II, III, or IV, e.g., a fumaric acidderivative (e.g., DMF or MMF).

In some embodiments method 4 provides a method of slowing or preventingneurodegeneration in a patient in need thereof, by administering thecompound in an amount and for a period of time sufficient to slow orprevent demyelination, axonal loss, and/or neuronal death, e.g., by atleast 30% relative to a control.

In some embodiments method 5 comprises:

-   -   a) administering to the mammal a therapeutically effective        amount of at least one first compound that upregulates the Nrf2        pathway, and    -   b) administering a therapeutically effective amount of at least        one second compound that does not upregulate the Nrf2 pathway.

In some embodiments of method 5, the at least one first compound, usedin step (a), is a compound of Formula I, II, III, or IV, e.g., a fumaricacid derivative (e.g., DMF or MMF); and the at least one secondcompound, which is used in step (b), is an immunosuppressive or animmunomodulatory compound that does not upregulate the Nrf2 pathway(e.g., by more than 30% over a control).

In some embodiments method 5 comprises administering to the mammal atherapeutically effective amount of a compound of Formula I, II, III, orIV.

In some embodiments of methods 1-5, the at least onecompound beingscreened, identified, evaluated, or used for treating a neurologicaldisorder is not fumaric acid or its salt, or a fumaric acid derivative(e.g., DMF or MMF).

Other features and embodiments of the invention will be apparent fromthe following description and the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 demonstrates that DMF and MMF are activators of Nrf2 atconcentrations within clinical exposure range (cells in culture).

FIG. 2 shows results of RNAi experiments.

FIG. 3 shows evidence of Nrf2 activation by DMF and MMF In vivo.

FIG. 4 shows evidence of Nrf2 activation by DMF and MMF In vivo.

Fumaric acid esters, such as DMF, have been proposed for treatment of MS(see, e.g., Schimrigk et al., Eur. J. Neurol., 2006, 13(6):604-10; DrugsR&D, 2005, 6(4):229-30).

Provided are, among other things, means for identifying compounds with anew therapeutic modality useful in at least one of multiple neurologicalindications and, optionally, complementary to other drugs for thetreatment of a neurological disease, including a number of currentlyused immunomodulators.

DMF is a member of a large group of anti-oxidant molecules known fortheir cytoprotective and anti-inflammatory properties. These moleculesalso share the property of the Nrf2 pathway activation. Thus, thefinding that DMF activates the Nrf2 pathway in conjunction with theneuroprotective effects of DMF further offers a rationale foridentification of structurally and/or mechanistically related moleculesthat would be expected to be therapeutically effective for the treatmentof neurological disorders, such as, e.g., MS.

Certain terms are defined in this section; additional definitions areprovided throughout the description.

The terms “activation” and “upregulation,” when used in reference to theNrf2 pathway, are used interchangeably herein.

The terms “disease” and “disorder” are used interchangeably herein.

The term “a drug for treating a neurological disease” refers to acompound that has a therapeutic benefit in a specified neurologicaldisease as shown in at least one animal model of a neurological diseaseor in human clinical trials for the treatment of a neurological disease.

The term “neuroprotection” and its cognates refer to prevention or aslowing in neuronal degeneration, including, for example, demyelinationand/or axonal loss, and/or, neuronal and/or oligodendrocyte death.Neuroprotection may occur through several mechanisms, e.g., throughreducing inflammation, providing neurotrophic factors, scavenging freeradicals, etc. As used herein, a compound is considered neuroprotectiveif it (1) upregulates the Nrf2 pathway above a certain threshold and (2)provides neuroprotection, regardless of possible other mechanisms ofaction.

The terms “treatment,” “therapeutic method,” “therapeutic benefits,” andthe like refer to therapeutic as well as prophylactic/preventativemeasures. Thus, those in need of treatment may include individualsalready having a specified disease and those who are at risk foracquiring that disease.

The terms “therapeutically effective dose” and “therapeuticallyeffective amount” refer to that amount of a compound which results in atleast one of prevention or delay of onset or amelioration of symptoms ofa neurological disorder in a subject or an attainment of a desiredbiological outcome, such as reduced neurodegeneration (e.g.,demyelination, axonal loss, and neuronal death) or reduced inflammationof the cells of the CNS.

In one aspect, provided are methods of evaluating neuroprotectiveproperties of test compounds, including the following methods:

-   -   1) methods of screening for new candidate compounds that may be        useful for treating a neurological disease;    -   2) methods of evaluating neuroprotective properties of drugs and        candidates that are used or proposed for treating a neurological        disease;    -   3) methods of comparing (e.g., for bioequivalence) two or more        pharmaceutical compositions which contain fumaric acid        derivatives;

In some embodiments, methods 1-3 may comprise:

-   -   a) contacting a cell with the test compound,    -   b) determining whether the Nrf2 pathway is upregulated in the        cell,        and, in some embodiments, additionally performing the following        step(s):    -   c) determining whether the test compound slows or prevents        demyelination, axonal loss, and/or neuronal death, and/or    -   d) selecting the test compound as a candidate for treating        neurodegeneration in a neurological disease if 1) the Nrf2        pathway is upregulated and 2) demyelination, axonal loss, and/or        neuronal death are/is prevented or slowed.

Method 1

In some embodiments the methods of screening for a candidate compoundfor treating a neurological disease comprise:

-   -   a) contacting a cell with a plurality of test compounds,    -   b) determining whether the Nrf2 pathway is upregulated in the        cell, and    -   c) selecting from the plurality of compounds at least one        compound that upregulates the Nrf2 pathway,        wherein an upregulation of the Nrf2 pathway by the selected at        least one compound indicates that the selected at least one        compound may be useful for treating a neurological disease. For        example, the plurality of compounds may be represented by a        combinatorial chemical library, and the screening method may be        performed by a high-throughput screening as described in, e.g.,        High-Throughput Screening in Drug Discovery (Methods and        Principles in Medicinal Chemistry), by Jörg Hüser (ed.), John        Wiley & Sons (2006).

Combinatorial libraries of compounds are also described in, e.g.,Solid-Supported Combinatorial and Parallel Synthesis ofSmall-Molecular-Weight Compound Libraries (Tetrahedron OrganicChemistry) Ian Salusbury (ed.), Elsevier (1998); CombinatorialLibraries: Synthesis, Screening and Application Potential (LibraryBinding), by Riccardo Cortese (ed.), Walter de Gruyter (1995). Thelibraries of compounds may be, for example, quinone libraries and otherlibraries as described in Mittoo, Comb. Chem. & High Throughput Screen,2006, 9:421-423.

In some embodiments, the at least one compound or plurality of compoundsbeing screened and/or selected comprises at least one compound selectedfrom at least one of the following groups of compounds: mild alkylatingagents, Michael addition acceptors or compounds that are metabolized toMichael addition acceptors, including compounds of Formulas I, II, III,or IV.

In some of the embodiments, the at least one compound is selected fromfumaric acid, its salts, and fumaric acid derivatives.

Method 2

Also provided are methods of evaluating neuroprotective properties of atleast one drug or drug candidate for treating at least one neurologicaldisease. Such methods comprise:

a) contacting a cell with the at least one drug or drug candidate, and

b) determining whether the Nrf2 pathway is upregulated in the cell,

NAI-1500508785v1 wherein the upregulation of the Nrf2 pathway by the atleast one drug or drug candidate indicates that the at least one drug ordrug candidate is neuroprotective in treating a human having aneurological disease.

In some embodiments, the upregulation of the Nrf2 pathway by the atleast one drug or drug candidate indicates that the at least one drug ordrug candidate has at least one activity selected from slowingdemyelination, slowing the loss of axons, and slowing the rate ofneuronal death.

In some embodiments, the method of evaluating at least one drug or drugcandidate comprises an additional step:

c) evaluating demyelination, loss of axons, and/or neuronal death.

In some embodiments, steps a) and c) are performed in vivo in at leastone model of a neurological disease, e.g., as described below.

In other embodiments, particularly those in which the neurologicaldisease is multiple sclerosis or another demyelinating disease, theevaluated at least one drug or drug candidate for a neurological diseaseis chosen from the following: FTY720(2-(4-octylphenethyl)-2-aminopropane-1,3-diol; Novartis); anti-IL12antibody (e.g., ABT-874; Abbott Laboratories); GSK683699 (GSK/Tanabe);NeuroVax (Immune Response Corp.; Darlington, Curr. Opin. Mol. Ther.,2005, 7(6):598-603); anti-CCR2 antibody (e.g., MLN 1202; Millennium);interferon β-1a (e.g., Avonex®; Biogen Idec); anti-α4-integrin antibody(e.g., Tysabri®; Biogen Idec/Elan); anti-CD20 antibody (e.g., Rituxan®(Biogen Idec/Genentech); TV 5010 (Teva); NBI-788 (Neurocrine); MBP8298(BioMS (see Warren et al., Eur. J. Neurol., 2006, 13(8):887-95); Mylinax(Oral Cladribine; 2-chlorodeoxyadenosine; Serono/IVAX); Teriflunomide((Z)-2-cyano-N-(4-(trifluoromethyl)phenyl)-3-hydroxybut-2-enamide;Sanofi-Aventis); Temsirolimus (Wyeth); Laquinimod(5-chloro-N-ethyl-1,2-dihydro-4-hydroxy-1-methyl-2-oxo-N-phenylquinoline-3-carboxamide;Active Biotech/Teva); and interferon tau (Tauferon; Pepgen).

In some embodiments, the at least one drug or drug candidate beingevaluated is at least one compound selected from at least one classselected from a mild alkylating agent, a Michael addition acceptor, anda compound that is metabolized to a Michael addition acceptor, includingcompounds of Formulas I, II, III, or IV.

In some of the embodiments, the compound is fumaric acid, its salt, or afumaric acid derivative.

Method 3

Also provided are methods of comparing (e.g., for bioequivalence) atleast two pharmaceutical compositions. Such methods comprise:

-   -   a) contacting a cell with at least one first composition        comprising a test compound, and    -   b) comparing the level of the Nrf2 pathway upregulation in the        cell by the test compound to the corresponding level of the Nrf2        pathway upregulation in a cell treated with at least one second        composition (“comparator composition”) comprising DMF, MMF, or        both.

In some embodiments, substantially dissimilar levels of upregulation bythe at least one first and at least one second compositions indicatethat the compositions are not bioequivalent.

In some embodiments, the test compound is fumaric acid, its saltthereof, a fumaric acid derivative, or mixtures thereof. In someembodiments, the first composition comprises at least one of DMF, MMF,and both DMF and MMF. In some embodiments, the dose and/or theformulation of the at least one first composition differs from the doseand/or the formulation of the at least one second composition. The atleast one first composition may be a controlled release composition suchas, e.g., compositions described in WO 2006/037342.

In some embodiments, the method further comprises and additional step:

-   -   c) comparing at least one pharmacokinetic parameter of the at        least one first and the at least one second compositions.

Pharmacokinetic parameters and methods for evaluating the same are wellknown and are described in, e.g., Pharmacokinetics, Second Edition(Drugs and the Pharmaceutical Sciences) by Milo Gibaldi et al. (eds.),Informa Healthcare (1982). Examples of such pharmacokinetic parametersthat can be evaluated include serum half-life, clearance, and volumedistribution.

In some embodiments, substantially dissimilar pharmacokineticparameter(s) of the a least one first and at least one secondcompositions indicate that the compositions are not bioequivalent.

In some embodiments, the test compound being evaluated is a mildalkylating agent, and more specifically, a Michael addition acceptor, ora compound that is metabolized to a Michael addition acceptor.

In some of the embodiments, the test compound is fumaric acid or itssalt, or a fumaric acid derivative.

Also provided are methods of treating a mammal who has or is at risk fordeveloping a neurological disease, including the following methods:

-   -   4) methods of treating a neurological disease by administering        to the subject in need thereof at least one compound that is        partially structurally similar to DMF or MMF (including        compounds selected using methods 1-3 described above); and    -   5) methods of treating a neurological disorder by a combination        therapy that includes administration of a first compound that        does not upregulate the Nrf2 pathway and a second compound that        upregulates the Nrf2 pathway.

Method 4

Also provided are methods of treating a neurological disease byadministering to the subject in need thereof at least one compound thatis at least partially structurally similar to DMF and/or MMF.

In some embodiments of method 4, a method of treating a mammal who hasor is at risk for a neurological disease is provided. The methodscomprises administering to the mammal a therapeutically effective amountof at least one neuroprotective compound which has Formula I, II, III,or IV, e.g., a fumaric acid derivative (e.g., DMF or MMF).

In some embodiments of method 4, a method of slowing or preventingneurodegeneration (more specifically, e.g., demyelination, axonal loss,and/or neuronal death) in a subject in need thereof, by administeringthe at least one compound in an amount and for a period of timesufficient to do at least one of slow or prevent demyelination, slow orprevent axonal loss, and alow or prevent neuronal death, e.g., by atleast 30%, 50%, 100% or higher over a control over a period of at least5, 10, 12, 20, 40, 52, 100, or 200 weeks, or more.

Method 5

Also provided are methods of treating a mammal having a neurologicaldisease by combination therapy. In some embodiments such methodscomprise:

-   -   a) administering to the mammal a therapeutically effective        amount of at least one first compound that upregulates the Nrf2        pathway, and    -   b) administering a therapeutically effective amount of at least        one second compound that does not upregulate the Nrf2 pathway.

In some of embodiments of method 5, the at least one first compound,used in step (a), is a compound of Formula I, II, III, or IV, e.g., DMFor MMF; and the at least one second compound, which is used in step (b),is an immunosuppressive or an immunomodulatory compound that does notupregulate the Nrf2 pathway (e.g., by more than 30%, 50%, 100% over acontrol).

In some embodiments of method 5, the method comprises administering tothe mammal a therapeutically effective amount of a compound of FormulaI, II, III, or IV.

In method 5, the at least one first compound and the at least one secondcompound may be administered concurrently (as separate compositions or amixed composition) or consecutively over overlapping or non-overlappingintervals. In the sequential administration, the at least one firstcompound and the at least one second compound can be administered in anyorder. In some embodiments, the length of an overlapping interval ismore than 2, 4, 6, 12, 24, or 48 weeks, for example.

Michael addition acceptors generally include olefins or acetylenesconjugated to an electron withdrawing group, such as carbonyl containinggroups, thiocarbonyl containing groups, cyano, sulfonyl, sulfonamido,amido, formyl, keto, and nitro. Exemplary carbonyl groups includecarboxylic acid esters and carboxylic acid.

In some embodiments of methods 1-5, the at least one compound beingscreened, identified, evaluated, or used for treating a neurologicaldisorder is selected from a mild alkylating agent, aMichael additionacceptor, and a compound that is metabolized to a Michael additionacceptor.

In some embodiments, the Michael addition acceptor has the structure ofFormula I:

or a pharmaceutically acceptable salt thereof, wherein:

X is O; S; C(R)(C₁₋₁₂)alkyl; or C(R)(C₂₋₁₂)alkenyl, wherein R is H,(C₁₋₁₂)alkyl or (C₂₋₁₂)alkenyl;

R¹, R², R³ and R⁴ are independently selected from: H; OH; O⁻; CO₂H, CO₂⁻; SH; S⁻; SO₂H, SO₂ ⁻; (C₁₋₂₄)alkyl; (C₁₋₂₄)alkenyl; (C₆₋₅₀)aryl,CO₂(C₁₋₂₄)alkyl; SO₂(C₁₋₂₄)alkyl; CO₂(C₁₋₂₄)alkenyl; SO₂(C₁₋₂₄)alkenyl;CO₂Y, wherein Y is psoralen-9-yl, retinyl, alpha-tocopherol, calciferyl,corticosteroid-21-yl or monosaccarid-ω-yl; (C₁₋₂₄)alkoxy;(C₁₋₂₄)alkenyloxy; (C₆₋₅₀)aryloxy; (C₁₋₂₄)alkylthio; (C₁₋₂₄)alkenylthio;(C₆₋₅₀)arylthio, amino; amido; arylalkyl; cyano; nitro; sulfonyl;sulfoxido; sulfonamido; formyl; keto; and D and L natural or unnaturalamino acids; or any two of X, R¹, R² and R³, and R⁴ may be joinedtogether to form a cyclic moiety; and wherein the alkyl, alkoxy,alkenyl, alkenyloxy, aryl and aryloxy groups may be optionallysubstituted with at least one group chosen from halogen (F, Cl, Br, orI), OH, (C₁₋₄)alkoxy, nitro and cyano.

In some embodiments, the at least one Michael addition acceptor has thestructure of Formula I, with the following provisos:

R¹ is selected from: H; OH; O⁻; CO₂H, CO₂ ⁻; SH; S⁻; SO₂H, SO₂ ⁻;(C₁₋₂₄)alkyl; (C₁₋₂₄)alkenyl; (C₆₋₅₀)aryl; CO₂(C₁₋₂₄)alkyl;SO₂(C₁₋₂₄)alkyl; CO₂(C₁₋₂₄)alkenyl; SO₂(C₁₋₂₄)alkenyl; CO₂Y, wherein Yis psoralen-9-yl, retinyl, alpha-tocopherol, calciferyl,corticosteroid-21-yl or monosaccarid-ω-yl; (C₁₋₂₄)alkoxy;(C₁₋₂₄)alkenyloxy; (C₆₋₅₀)aryloxy; (C₁₋₂₄)alkylthio; (C₁₋₂₄)alkenylthio;(C₆₋₅₀)arylthio; arylalkyl; amino; amido; cyano; nitro; sulfonyl,sulfoxido; sulfonamido; formyl, keto; and D or L natural or unnaturalamino acids; and wherein the alkyl, alkoxy, alkenyl, alkyenyloxy, aryland aryloxy groups may be optionally substituted with at least one groupchosen from halogen (F, Cl, Br, or I), OH, (C₁₋₄)alkoxy, nitro andcyano;

R² is selected from: H; CO₂H; CO₂ ⁻; SO₂H; SO₂ ⁻; (C₁₋₂₄)alkyl;(C₁₋₂₄)alkenyl; (C₆₋₅₀)aryl; CO₂(C₁₋₂₄)alkyl; SO₂(C₁₋₂₄)alkyl;CO₂(C₁₋₂₄)alkenyl; SO₂(C₁₋₂₄)alkenyl; CO₂Y, wherein Y is psoralen-9-yl,retinyl, alpha-tocopherol, calciferyl, corticosteroid-21-yl ormonosaccarid-ω-yl; (C₁₋₂₄)alkoxy; (C₁₋₂₄)alkenyloxy; (C₆₋₅₀)aryloxy;(C₁₋₂₄)alkylthio; (C₁₋₂₄)alkenylthio; (C₆₋₅₀)arylthio, amido; arylalkyl;cyano; nitro; sulfonyl, sulfoxido, sulfonamido; formyl, keto; and D or Lnatural or unnatural amino acids; wherein the alkyl, alkoxy, alkenyl,alkenyloxy, aryl and aryloxy groups may be optionally substituted withat least one group chosen from halogen (F, Cl, Br, or I), OH,(C₁₋₄)alkoxy, nitro and cyano; and

R³ and R⁴ are independently selected from: H; CO₂H; CO₂ ⁻; SO₂H; SO₂ ⁻;(C₁₋₂₄)alkyl; (C₁₋₂₄)alkenyl; (C₆₋₅₀)aryl; CO₂(C₁₋₂₄)alkyl;SO₂(C₁₋₂₄)alkyl; CO₂(C₁₋₂₄)alkenyl; SO₂(C₁₋₂₄)alkenyl; CO₂Y, wherein Yis psoralen-9-yl, retinyl, alpha-tocopherol, calciferyl,corticosteroid-21-yl or monosaccarid-ω-yl; (C₁₋₂₄)alkoxy;(C₁₋₂₄)alkenyloxy; (C₆₋₅₀)aryloxy; (C₁₋₂₄)alkylthio; (C₁₋₂₄)alkenylthio;(C₆₋₅₀)arylthio; amido; arylalkyl; cyano; nitro; cyano; nitro; sulfonyl;sulfoxido; sulfonamido; formyl; and keto; wherein the alkyl, alkoxy,alkenyl, alkenyloxy, aryl and aryloxy groups may be optionallysubstituted with at least one group chosen from halogen (F, Cl, Br, orI), OH, (C₁₋₄)alkoxy, nitro and cyano.

In some embodiments, the at least one Michael addition acceptor has thestructure of Formula II:

or a pharmaceutically acceptable salt thereof, wherein:

X is selected from O; S; C(R)(C₁₋₁₂)alkyl; and C(R)(C₂₋₁₂)alkenyl,wherein R is selected from H; (C₁₋₁₂)alkyl; and (C₂₋₁₂)alkenyl; and R¹,R², R³, and R⁴ are independently selected from: H; OH; O⁻; CO₂H; CO₂ ⁻;(C₁₋₁₂)alkyl; (C₁₋₁₂)alkenyl; and CO₂(C₁₋₁₂)alkyl;

or any two of X, R¹, R² and R³ may be joined together to form a cyclicmoiety.

In some embodiments of the compounds of Formulae I-IV, thepharmaceutically acceptable salt is a salt of a metal (M) cation,wherein M can be an alkali, alkaline earth, or transition metal such asLi, Na, K, Ca, Zn, Sr, Mg, Fe, or Mn.

In some embodiments of methods 1-5, the compounds of Formula I includefumaric acid, its salts, and fumaric acid derivatives.

In some embodiments, the at least one compound of Formula I has thestructure of Formula III:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ and R³ are independently selected from OH; O⁻; (C₁₋₂₄)alkoxy;(C₁₋₂₄)alkenyloxy; (C₆₋₅₀)aryloxy; psoralen-9-yloxy; retinyloxy;alpha-tocopheroloxy; calciferyloxy; corticostreoid-21-yloxy;monosaccarid-ω-yloxy; amino; and a D or L natural or unnatural aminoacid; and wherein at least one of the (C₁₋₂₄)alkoxy; (C₁₋₂₄)alkenyloxy;and (C₆₋₅₀)aryloxy groups may be optionally substituted with at leastone group chosen from halogen (F, Cl, Br, or I), OH, (C₁₋₄)alkoxy, nitroand cyano.

Compounds wherein at least one of R¹ and R³ is derived from a natural orunnatural D or L amino acid are described in U.S. application Ser. No.10/433,295, paragraphs 10 to 11 and 18-28, and Ser. No. 11/421,083,which are incorporated herein by reference.

In some embodiments, the compound of formula (I) has the structure ofFormula IV:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ and R³ are independently selected from OH; O⁻; (C₁₋₂₄)alkoxy;allyloxy; vinyloxy; (C₆₋₅₀)aryloxy; psoralen-9-yloxy; retinyloxy;alpha-tocopheroloxy; calciferyloxy; corticostreoid-21-yloxy;monosaccarid-ω-yloxy; amino; and a D or L natural or unnatural aminoacid; and wherein at least one of the the (C₁₋₂₄)alkoxy, allyloxy,vinyloxy, and (C₆₋₅₀)aryloxy may be optionally substituted with at leastone group chosen from Cl, F, I, Br, OH, (C₁₋₄)alkoxy, nitro, and cyano.

In some embodiments, the “fumaric acid derivative” is chosen from thecompounds of Formula III, compounds of Formula IV and the following:

1) fumaric acid amides derived from natural and unnatural amino D or Lacids, as described in U.S. patent application Ser. No. 10/433,295,paragraphs 10 to 11 and 18-28, and Ser. No. 11/421,083.

2) a carbocyclic or oxacyclic fumaric acid oligomer as described in U.S.patent application Ser. No. 10/511,564, paragraphs 15-44; and

-   -   3) a glycerol or alkane diol or polyol derivative of fumaric        acid as described in U.S. Pat. Nos. 4,851,439, 5,149,695,        5,451,667, at cols. 2-4.

In some embodiments, “fumaric acid derivative” is one or more dialkylfumarates (e.g., DMF), mono alkyl fumarates (MMF) or salts thereof.

In some of the embodiments of methods 1-5, the at least one compoundbeing screened, evaluated, compared or used for treating a neurologicaldisorder is not fumaric acid or its salt, or a fumaric acid derivative(e.g., DMF or MMF).

Nrf2 (Nuclear Factor-E2-related factor 2; for sequence of the Nrf2, seeAccession No. AAB32188) is a transcription factor that, upon activationby oxidative stress, binds to the antioxidant response element (ARE),and activates transcription of Nrf2-regulated genes. This pathway hasbeen well characterized for its role in hepatic detoxification andchemoprevention through the activation of phase II gene expression.ARE-regulated genes may also contribute to the maintenance of redoxhomeostasis by serving as endogenous anti-oxidant systems. At present,the list of Nrf2-regulated genes contains over 200 genes encodingproteins and enzymes involved in detoxification and antioxidant response(Kwak et al., J. Biol. Chem., 2003, 278:8135) such as, e.g., HO-1,ferritin, glutathione peroxidase, glutathione-S-transferases (GSTs),NAD(P)H:quinone oxidoreductases, now commonly known as nicotinamidequinone oxidoreductase 1 (NQO1; EC 1.6.99.2; also known as DT diaphoraseand menadione reductase), NQO2, g-glutamylcysteine synthase (g-GCS),glucuronosyltransferase, ferritin, and heme oxygenase-1 (HO-1), as wellas any one of the enzymes proteins listed in Table 1 in Chen & Kunsch,Curr. Pharm. Designs, 2004, 10:879-891; Lee et al., J. Biol. Chem.,2003, 278(14):12029-38, and Kwak, supra.

Accordingly, in some embodiments, the at least one Nrf2-regulated genewhich is used to assess the activation of the Nrf2 pathway is selectedfrom a phase II detoxification enzyme, an anti-oxidant enzyme, an enzymeof the NADPH generating system, and Nrf2 itself. Examples of the phaseII detoxification enzymes include NQO1, NQO2, GST-Ya, GST-pi, GST-theta2, GST-mu (1,2,3), microsomal GST 3, catalytic y-GCS, regulatory-GCS,microsomal epoxide hydrolase, UDP-glucuronosyltransferase,transaldolase, transketolase, and drug-metabolizing enzyme. Examples ofthe anti-oxidant enzymes include HO-1, ferritin (L), glutathionereductase, glutathione peroxidase, metallothionein I, thioredoxin,thioredoxin reductase, peroxiredoxin MSP23, Cu/Zn superoxide dismutase,and catalase. Examples of the enzymes of the NADPH generating systeminclude malic enzyme, UDP-glucose dehydrogenase, malate oxidoreductase,and glucose-6-phosphate dehydrogenase.

The antioxidant response element (ARE, also referred to as theelectrophile response element (EpRE), GRE1, ARE4, and StREb) is acis-acting DNA regulatory element with a core nucleotide sequence of5′-TGA(C/T/G)NNNGC-3′ (SEQ ID NO:1) (Rushmore et al., J. Biol. Chem.,1991, 266(18):11632-9; see also Nioi et al., Mutation Res., 2004,555:14-171).

Accordingly, in some embodiments, the DNA sequence of the ARE element,to which Nrf2 binds (whether the former is a part of an endogenous geneor an artificial construct), comprises the core ARE sequenceTGA(C/T/G)NNNGC (SEQ ID NO:2) or the ARE consensus sequence(G/A)TGA(C/T/G)NNNGC(A/G) (SEQ ID NO:3). In further specificembodiments, the ARE sequence comprises any one of the “minimalenhancer” sequences shown in Table 1.

In some embodiments, the ARE sequence further comprises at least one ofcorresponding 5′- and 3′-USR sequences as shown in Table 1. In someembodiments, the ARE sequence comprises the sequence GTGANNNNGCA (SEQ IDNO:4), or more particularly, the mouse (NNNN=gtcg) or human (NNNN=ctca)versions thereof.

TABLE 1 Minimal Species Gene Element 5′-USR enhancer 3′-USR SEQ ID NOmouse nqo1 ARE agTCAca GTGAgtcgGCA aaattt SEQ ID NO: 5 rat NQO1 AREagTCAca GTGACttgGCA aaatct SEQ ID NO: 6 human NQO1 ARE agTCAcaGTGACtcaGCA gaatct SEQ ID NO: 7 mouse gsta1 EpRE gcTAAtg GTGACaaaGCAactttc SEQ ID NO: 8 rat GSTA2 ARE gcTAAtg GTGACaaaGCA actttcSEQ ID NO: 9 mouse gsta3 ARE ctcAggc ATGACattGCA tttttc SEQ ID NO: 10rat GSTP1 GPE1 agTCAct ATGATtcaGCA acaaaa SEQ ID NO: 11 human GCLC ARE4ccTCccc GTGACtcaGCG ctttgt SEQ ID NO: 12 human GCLM EpRE gaagAcaATGACtaaGCA gaaatc SEQ ID NO: 13 mouse ho1 StREb cccAAcc ATGACacaGCAtaaaag SEQ ID NO: 14 ARE ′core′ . . .  TGACnnnGC SEQ ID NO: 15ARE consensus . . . TAAnn ATGACnnnGCA aaaa SED ID NO: 16  C G   T     Gtttt

A current model of Nrf2 function is as follows. Under basal conditions,Nrf2 is sequestered in the cytoplasm to the actin-bound Kelch-likeECH-associated protein 1 (Keap1; Accession No. NP_(—)987096 for humanKeap1), a Cullin3 ubiquitin ligase adaptor protein. More specifically,the N-terminal domain of Nrf2, known as Neh2 domain, is thought tointeract with the C-terminal Kelch-like domain of Keap1. In response toxenobiotics or oxidative stress, Nrf2 is released from the Keap1/Nrf2complex, thereby promoting nuclear translocation of Nrf2 and concomitantactivation of ARE-mediated gene transcription. Keap1 function, in turn,requires association with Cullin3, a scaffold protein that positionsKeap1 and its substrate in proximity to the E3 ligase Rbx1, allowing thesubstrate (Nrf2) to be polyubiquitinated and thus targeted fordegradation. The exact mechanism of how the Keap1/Nrf2 complex sensesoxidative stress is not fully understood. Human Keap1 contains 25cysteine residues that were hypothesized to function as sensors ofoxidative stress; 9 of the cysteines are thought to be highly reactive(Dinkova-Kostova et al., PNAS, 2005, 102(12):4584-9). It was theorizedbut is not relied on for the purposes of this invention that alkylationof cysteins leads to a conformational change, resulting in theliberation of Nrf2 from Nrf2/Keap1/Cullin3 complexes, followed bynuclear translocation of the liberated Nrf2.

In some embodiments, methods 1-3 described herein comprise contacting acell with at least one test compound and determining whether the Nrf2pathway is upregulated in the cell. In such methods, an upregulation ofthe Nrf2 pathway above a threshold (e.g., by at least 30%, 50%, 100%,200%, 500% over a control) indicates that the at least one compound hascertain biological properties beneficial in treating a neurologicaldisease (e.g., neuroprotective properties).

The ability of a compound to activate the Nrf2 pathway can be determinedby one or more in vitro and in vivo assays, including, e.g., thefollowing assays described below.

i) Expression Levels of Nrf2—

The sequence of the promoter region of the nrf2 gene (−1065 to −35) hasbeen published, for example, in Chan et al., PNAS, 1996, 93:13943-13948.One may use an artificially constructed expression construct containingthe Nrf2 promoter element and an artificial reporter gene.Alternatively, one may use PCR or Northern blotting to determineexpression levels of Nrf2 mRNA, or Western blotting to determine Nrf2protein levels. Exemplary procedures for determining expression levelsof Nrf2 are described in Kwak et al., Mol. Cell. Biol. 2002,22(9):2883-2892 and Kwak et al., Mol. Med., 2001, 7:135-145. Antibodiesagainst Nrf2 are can be produced by methods known in the art and arecommercially available from, for example, StressGen. Accordingly, insome embodiments, the Nrf2 pathway is activated so that the expressionlevels of Nrf2 are increased by, for example, at least 30%, 50%, 100%,200%, 500% or more as compared to the non-activated state.

ii) Subcellular Localization and/or Nuclear Translocation of Nrf2—

Such assays include cell staining, or analysis of cytoplasmic versusnuclear cell extracts.

For example, a Nrf2-green fluorescence protein (GFP) fusion proteinconstruct can be made and introduced into cells and visualized asdescribed in, e.g., Kraft et al., J. Neurosci., 2004, 24, 1101-1112; andSatoh et al., PNAS, 2006, 103(3):768-773. Accordingly, in someembodiments, the Nrf2 pathway is activated so that the ratio betweencytoplasmic and nuclear Nrf2 is elevated by, for example, at least 30%,50%, 100%, 200%, 500% or more as compared to the non-activated state.

iii) Expression Levels and/or Activity of One or More Genes Under theControl of Nrf2—

Such genes under the control of Nrf2 include endogenous or artificiallyintroduced reporter genes in reporter constructs introduced into cells.For example, expression levels of endogenous or exogenously introducedNQO1 may be determined as described in the Examples. Alternatively, areporter gene construct with one or more ARE sites operably linked to areporter gene (e.g., luciferase or GFP) can be made, as described in,e.g., Satoh et al., PNAS, 2006, 103(3):768-773. Expression levels of anNrf-2 induced gene product can be measured at the protein (e.g., byWestern blotting or enzymatic activity assays) or at the mRNA levels(e.g., by PCR). Methods for performing RT-PCT are described in, e.g.,Calabrese et al., J. Neurosci. Res., 2005, 79:509-521 for HO-1, inWierinckx et al., J. Neuroimmunology, 2005, 166:132-143 for NQO1.Methods for measuring enzymatic activity of NQO1, using for example,menadione as a substrate, are described in Dinkova-Kostova et al., PNAS,2001, 98:3404-09 or by Prochaska et al., Anal. Biochem., 1988,169:328-336. Methods for measuring GST activity, using for example,1-chloro-2,4-dinitrobenzene as a substrate, are described in Ramos-Gomezet al., J. Neurosci., 2004, 24(5):1101-1112 and Habig et al., 1974, J.Biol. Chem., 219, 7130-7139. Methods for measuring HO-1 activity aredescribed in, e.g., in Calabrese et al., 2005, J. Neurosci. Res.,79:509-521. Accordingly, in some embodiments, the Nrf2 pathway isactivated so that the expression levels and/or activity of the geneproduced are increased by, for example, at least 30%, 50%, 100%, 200%,500% or more as compared to the non-activated state.

iv) Levels of Nrf2 Binding to ARE—

For example, such assays may utilize electromobility shift assays (EMSA)and Chromatin Immununoprecipitation (ChIP) assay, as described in, e.g.,Satoh et al., PNAS, 2006, 103(3):768-773 and Kwak et al., Mol. CellBiol., 2002, 22(9):2883-2892. Accordingly, in some embodiments, the Nrf2pathway is activated so that the level of Nrf2 binding to ARE isincreased by, for example, at least 30%, 50%, 100%, 200%, 500% or moreas compared to the non-activated state.

v) The Stability of Nrf2/Keap1 Complexes—

Such assay may include analysis of immunoprecipitated complexes withNrf2 and/or Keap1 or other Nrf2/Keap1-associated proteins as describedin, e.g., Satoh et al., PNAS, 2006, 103(3):768-773. Anti-Keap1antibodies can be produced using methods known in the art and areavailable commercially from, for example, Santa Cruz Biotechnology.Accordingly, in some embodiments, the Nrf-2 pathway is activated so thatthe stability of Nrf2/Keap1 complexes is increased by, for example, atleast 30%, 50%, 100%, 200%, 500% or more as compared to thenon-activated state.

vi) Modification (e.g., Alkylation Levels) of Keap1 and OtherNrf2/Keap1-Associated Proteins—

Such assays may include mass spectrometric analysis ofimmunoprecipitated Keap1, using techniques as described in, e.g.,Dinkova-Kostova et al., PNAS, 2005, 102(12):4584-9 and Gao et al., J.Biol. Chem., on-line pub. Manuscript M607622200. In some embodiments,the Nrf-2 pathway is activated so that the level of Keap1 and otherNrf2/Keap1-associated proteins is increased by, for example, at least30%, 50%, 100%, 200%, 500% or more as compared to the non-activatedstate.

Alkylating capacity of a compound can be assessed using recombinantKeap1, by a competition reaction with 5,5′-dithiobis(2-nitrobenzoicacid) (DTNB) as described in, e.g., Gao et al., J. Biol. Chem., on-linepub. Manuscript M607622200.

In some embodiments, the cell being contacted with at least one testcompound is a neuron or a neuronal cell line. In some embodiments, thecell being contacted with the at least one test compound is selectedfrom a colon carcinoma cell line (e.g., DLD1), a neuroblastoma cell line(e.g., SkNSH or IMR32), and a primary monocyte. The cell may be a cellin culture (in vitro) or be inside of an animal (in vivo).

Cell viability, and in particular, neuronal viability can be assessed invivo or in vitro using any suitable method, including methods asdescribed in the Examples. For example, neuronal viability can beassessed using an MTT assay after exposure of neuronal cell cultures tocytotoxic levels of glutamate as described in, e.g., Shih et al., J.Neurosci., 2005, 25(44):10321-35. Additionally, cell viability may alsobe assessed in assays in which cell death is induced by oxidativedamage, for example, by the addition of glucose oxidase to astrocytecell cultures, as described in, e.g., Calabrese et al., J. Neurosci.Res., 2005, 79:509-521. In vivo assays may be performed as described in,e.g., Misgeld, Histochem. Cell Biol., 2005, 124:189-196.

The amount of the reporter gene expressed can be determined by anysuitable method. Expression levels, at the RNA or the protein level, canbe determined using routine methods. Expression levels are usuallyscaled and/or normalized per total amount of RNA or protein in thesample and/or a control, which is typically a housekeeping gene such asactin or GAPDH. RNA levels are determined by quantitative PCR (e.g.,RT-PCR), Northern blotting, or any other method for determining RNAlevels, e.g., as described in Cloning: A Laboratory Manual, by Sambrooket al. (eds.), 2nd ed., Cold Spring Harbor Laboratory Press, 1989; Lodieet al., Tissue Eng., 2002, 8(5):739-751); or as described in theExamples. Protein levels are determined using, Western blotting, ELISA,enzymatic activity assays, or any other method for determining proteinlevels as described in, e.g., Current Protocols in Molecular Biology, byAusubel et al. (eds.), John Wiley and Sons, 1998.

Expression levels may also be determined using reporter gene assays incell/tissue extracts or by tissue or whole-animal imaging. In additionto MRI, tissue imaging on living animals can be performed byfluorescence detection (Hoffman Lancet Oncol., 2002 3:546-556; Tung etal., Cancer Res., 2000, 60:4953-4958), bioluminescence detection (Shi etal., PNAS, 2001, 98:12754-12759; Luke et al., J. Virol., 2002,76:12149-12161; and U.S. Pat. Nos. 5,650,135 and 6,217,847), positronemission tomography (Liang et al., Mol. Ther., 2002, 6:73-82,near-infrared fluorescence (Tung et al., Cancer Res., 2000,60:4953-4958), or X-ray imaging (Hemminki et al., J. Nat. Cancer Inst.,2002, 94:741-749).

A neurological disease in methods 1-5 above can be a neurodegenerativedisease such as, for example, ALS, Parkinson's disease, Alzheimer'sdisease, and Huntington's disease. The neurological disease can also bemultiple sclerosis (MS), or other demyelinating diseases of the centralor peripheral nervous system. In some embodiments the form of MS inmethods 1-5 is selected from: relapsing remitting MS (RRMS), secondaryprogressive MS (SPMS), primary progressive MS (PPMS), and malignant MS(Marburg Variant).

The subject being treated or administered the compound as per methodsdescribed herein, is a mammal in need thereof, such as a subject in needof neuroprotection, including a subject who has or is at risk fordeveloping a demyelinating and another specified neurodegenerativedisease. The subject is mammalian, and can be a rodent or anotherlaboratory animal, e.g., a non-human primate. In some embodiments, thesubject is human.

Neurodegenerative diseases are described in, for example,Neurodegenerative Diseases: Neurobiology, Pathogenesis and Therapeutics,M. Flint Beal, Anthony E. Lang, Albert C. Ludolph, Cambridge UniversityPress (Jul. 11, 2005). Examples of neurological diseases suitable forthe methods described herein include neurodegenerative diseases such asamyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer'sdisease, and Huntington's disease. Other examples include demyelinatingneurological disease including, in addition to MS, the followingdiseases: acute haemorrhagic leucoencephalomyelitis, Hurst's disease,acute disseminated encephalomyelitis, optic neuritis, Devic's disease,spinal cord lesions, acute necrotizing myelitis, transverse myelitis,chronic progressive myelopathy, progressive multifocalleukoencephalopathy (PML), radiation myelopathy, HTLV-1 associatedmyelopathy, monophasic isolated demyelination, central pontinemyelinolysis, and leucodystrophy (e.g., adrenoleucodystrophy,metachromatic leucodystrophy, Krabbe's disease, Canavan's disease,Alexander's disease, Pelizaeus-Merbacher disease, vanishing white matterdisease, oculodentodigital syndrome, Zellweger's syndrome), chronicinflammatory demyelinating polyneuropathy (CIDP), acute inflammatorydemyelinating polyneuropathy (AIDP), Leber's optic atrophy, andCharcot-Marie-Tooth disease.

Additional examples of diseases suitable for the methods describedherein include polyneuritis and mitochondrial disorders withdemyelination. These disorders may be co-presented with, and possiblyaggravated by diabetes, e.g., insulin-dependent diabetes mellitus (IDDM;type I diabetes), or other diseases.

A test compound may be further assayed in an animal model of MS, knownas Experimental Autoimmune Encephalomyelitis (EAE) (Tuohy et al., J.Immunol., 1988, 141:1126-1130, Sobel et al. J. Immunol., 1984,132:2393-2401, and Traugott, Cell Immunol., 1989 119:114-129). Chronicrelapsing EAE provides a well established experimental model for testingagents that would be useful for the treatment of MS. The mouse EAE is aninduced autoimmune demyelinating disease with many similarities to humanMS in its clinical manifestations. In both EAE and MS, clinical diseaseis associated with blood-brain barrier (BBB) dysfunction, infiltrationof central nervous system by mononuclear cells (mainly macrophages and Tlymphocytes, and serum products), and demyelination (Baker et al. J.Neuroimmunol., 1990, 28:261; Butter et al., J. Neurol. Sci., 1991,104:9; Harris et al., Ann. Neurol., 1991, 29:548; Kermonde et al.,Brain, 1990, 113:1477).

Clinical signs of MS and demyelinating pathology in EAE result fromimmunization with CNS myelin proteins or peptides (e.g., MBP, PLP, andMOG) under Th1 conditions (direct immunization model), or by adoptivetransfer of CNS antigen-specific Th1 cells (adoptive transfer model)(Ben-Nun et al., Eur. J. Immunol., 1981, 11:195-199; Ando et al., CellImmunol., 1989, 124:132-143; Zamvil et al., Nature, 1985, 317:355-358;Zamvil et al., Ann. Rev. Immunol., 1990, 8:579-621). For example, in theSJL mouse model of EAE, immunization with the CNS peptide PLP 139-151 oradoptive transfer of PLP-specific Th1 cells results in a disease courseconsisting of an acute phase with loss of tail tone on day 10 to day 12,followed by hind limb paralysis and CNS mononuclear cell infiltration(Tuohy et al., J. Immunol., 1988, 141:1126-1130, Sobel et al., J.Immunol., 1984, 132:2393-2401, and Traugott, Cell Immunol., 1989,119:114-129). Resolution of clinical signs and recovery occurs on day 20to day 25 and the animals may undergo several more relapses less severethan the initial phase. EAE has been used to evaluate new therapeuticapproaches to T-cell-mediated autoimmune disease because of the clinicaland histopathological similarities to the human demyelinating MS.

The ability of a compound to slow or prevent neurodegeneration(including demyelination and neuronal death) can be assessed in the EAEmodel or another animal model, including for example, Thieler's murineencephalomyelitis virus (TMEV)-induced demyelinating disease, murinehepatitis virus (MHV), Semliki Forest Virus, or Sindbis virus asdescribed in, e.g., Ercoli et al., J. immunol., 2006, 175:3293-3298.

A compound may be optionally tested in at least one additional animalmodel (see, generally, Immunologic Defects in Laboratory Animals, eds.Gershwin et al., Plenum Press, 1981), for example, such as thefollowing: the SWR X NZB (SNF1) mouse model (Uner et al., J. AutoimmuneDisease, 1998, 11(3):233-240), the KRN transgenic mouse (K/BxN) model(Ji et al., Immunol. Rev., 1999, 69:139); NZB X NZW (B/W) mice, a modelfor SLE (Riemekasten et al., Arthritis Rheum., 2001, 44(10):2435-2445);the NOD mouse model of diabetes (Baxter et al., Autoimmunity, 1991,9(1):61-67), etc.); or mouse models of multiple sclerosis (see, e.g.,Linker et al., Eur. J. Immunol., 2002, 8(6):620-624, and Eugster et al.,Nat. Med., 1999, 29:626-632; and Gold et al., Brain, 2006,129:1953-1971).

Preliminary doses, for example, as determined in animal tests, and thescaling of dosages for human administration is performed according toart-accepted practices. Toxicity and therapeutic efficacy can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. In some embodiments compositions that exhibit largetherapeutic indices are used.

The therapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (i.e., theconcentration of the therapeutic compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture assays or animalmodels. Levels in plasma may be measured, for example, by ELISA or HPLC.The effects of any particular dosage can be monitored by a suitablebioassay. Examples of dosages are: about 0.1×10₅₀, about 0.5×10₅₀, about1×10₅₀, about 5×10₅₀, 10×10₅₀, about 50×10₅₀, and about 100×IC₅₀.

The data obtained from the in vitro assays or animal studies can be usedin formulating a range of dosages for use in humans. Therapeuticallyeffective dosages achieved in one animal model can be converted for usein another animal, including humans, using conversion factors known inthe art (see, e.g., Freireich et al., Cancer Chemother. Reports, 1966,50(4):219-244 and Table 2 for Equivalent Surface Area Dosage Factors).

TABLE 2 To: Mouse Rat Monkey Dog Human From: (20 g) (150 g) (3.5 kg) (8kg) (60 kg) Mouse 1 1/2 1/4 1/6  1/12 Rat 2 1 1/2 1/4 1/7 Monkey 4 2 13/5 1/3 Dog 6 4 3/5 1 1/2 Human 12 7 3 2 1

In some embodiments the dosage of such compounds lies within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. In some embodiments the dosage varies within this rangedepending upon the dosage form employed and the route of administrationutilized. Generally, a therapeutically effective amount may vary withthe subject's age, condition, and sex, as well as the severity of themedical condition in the subject. Examples of pharmaceuticallyacceptable dosages for compounds described herein are from 1 μg/kg to 25mg/kg, depending on the compounds, severity of the symptoms and theprogression of the disease. The appropriate therapeutically effectivedoses can be selected by a treating clinician and in some embodimentsrange approximately from 1 μg/kg to 20 mg/kg, from 1 μg/kg to 10 mg/kg,from 1 μg/kg to 1 mg/kg, from 10 μg/kg to 1 mg/kg, from 10 μg/kg to 100μg/kg, from 100 μg to 1 mg/kg. Additionally, certain specific dosagesare indicated in the Examples.

For DMF or MMF, an effective amount can range from 1 mg/kg to 50 mg/kg(e.g., from 2.5 mg/kg to 20 mg/kg or from 2.5 mg/kg to 15 mg/kg).Effective doses will also vary, as recognized by those skilled in theart, dependent on route of administration, excipient usage, and thepossibility of co-usage with other therapeutic treatments including useof other therapeutic agents. For example, an effective dose of DMF orMMR to be administered to a subject orally can be from about 0.1 g to 1g per pay, 200 mg to about 800 mg per day (e.g., from about 240 mg toabout 720 mg per day; or from about 480 mg to about 720 mg per day; orabout 720 mg per day). For example, the 720 mg per day may beadministered in separate administrations of 2, 3, 4, or 6 equal doses.

The dosage may be determined by a physician and adjusted, as necessary,to suit observed effects of the treatment. The compositions may be givenas a bolus dose, to maximize the circulating levels for the greatestlength of time after the dose. Continuous infusion may also be usedafter the bolus dose.

In some embodiments, compositions used in the methods described hereinfurther comprise a pharmaceutically acceptable excipient. As usedherein, the phrase “pharmaceutically acceptable excipient” refers to anyand all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, that are compatible with pharmaceutical administration. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. The compositions may also contain other activecompounds providing supplemental, additional, or enhanced therapeuticfunctions. The pharmaceutical compositions may also be included in acontainer, pack, or dispenser together with instructions foradministration.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Methods to accomplish theadministration are known in the art. “Administration” is not limited toany particular delivery system and may include, without limitation,parenteral (including subcutaneous, intravenous, intramedullary,intraarticular, intramuscular, or intraperitoneal injection), rectal,topical, transdermal, or oral (for example, in capsules (e.g., as,poweder, granules, microtablet, micropellets, etc.), suspensions, ortablets). Examples of some of formulations containing DMF and/or MMF aregiven in, e.g., U.S. Pat. Nos. 6,509,376, and 6,436,992.

Administration to an individual may occur in a single dose or in repeatadministrations, and in any of a variety of physiologically acceptablesalt forms, and/or with an acceptable pharmaceutical carrier and/oradditive as part of a pharmaceutical composition. Physiologicallyacceptable salt forms and standard pharmaceutical formulation techniquesand excipients are well known to persons skilled in the art.

The following Examples are intended for illustrative purposes and do notlimit the inventions claimed.

EXAMPLES Example 1

Human colon carcinoma DLD1 cells were treated with DMF or MMF atindicated concentrations (5, 15, or 50 μM) for 16 hours, rinsed withPBS, and harvested into reducing SDS sample buffer. The lysates weresubjected to SDS PAGE and the separated proteins wereelectrophoretically transferred onto nitrocellulose membranes forWestern blot analysis. To detect Nrf2 and NQO1, the membranes wereincubated with the respective primary antibodies overnight at 4° C.,washed, and incubated with peroxidase-conjugated secondary antibodiesfollowed by the chemiluminescent peroxidase substrate. Detection of thetarget protein band luminescence and image acquisition were done usingCCD-equipped imaging station Kodak2000R. The results shown in FIG. 1,demonstrate that DMF and MMF are potent activators of Nrf2 atconcentrations within clinical exposure range.

Example 2

DLD1 cells were grown in MEM supplemented with 10% fetal bovine serum.The cells were transfected with the indicated siRNA's using theLipofectamine reagent (Invitrogen) according to the manufacturer'sinstructions and 30 hrs later stimulated with 30 μM DMF for 40 hours.The cells were harvested and processed for Western blotting analysis ofNrf2 and NQO1 levels as described in Example 1. Sources and the identityof reagents used in Examples 1 and 2 are specified Table 3 below:

Target Reagent Source/Sequence Vendor Primary Nrf2 Nrf2 (T-19)goat polyclonal Santa Cruz Antibody antibody Biotechnology Keap1Keap1 (E- goat polyclonal Santa Cruz 20) antibody Biotechnology NQO1NQO1 mouse monoclonal Santa Cruz (A180) antibody Biotechnology GAPDHAnti- mouse monoclonal Ambion GAPDH antibody Secondary anti- HRP- sheepAmersham antibody mouse Mouse IgG Biosciences anti- HRP- donkey Amershamrabbit Rabbit IgG Biosciences anti-Goat HRP-Goat Bovine Santa Cruz IgGBiotechnology siRNA Nrf2 Nrf2-2 UCAUUGAACUGC Dharmacon UCUUUGGUU(antisense) (SEQ ID NO: 17) Keap1 Keap1-1 GAAUUAAGGCGG DharmaconUUUGUCCUU (antisense) (SEQ ID NO: 18)

The results are shown in FIG. 2 (for ease of representation, the imageof the Western blot is turned upside down). The results demonstrate thatDMF-induced upregulation of NQO1 requires Nrf2 and can be mimicked byactivation of Nrf2 through repression of Keap1. Therefore, DMF acts asan Nrf2 agonist causing cellular accumulation of Nrf2 and Nrf2 targetgene expression.

Example 3

For induction of EAE, mice received s.c. injections in the flanks andtail base of 50 μg MOG 35-55 peptide in PBS emulsified in an equalvolume of complete Freund's adjuvant (CFA) containing Mycobacteriumtuberculosis H37RA (Difco, Detroit Mich., USA) at a final concentrationof 0.5 mg/ml. Two injections of pertussis toxin (List BiologicalLaboratories Inc., California, USA; 200 ng per mouse i.p) were given ondays 0 and 2.

DMF and MMF was diluted in 200 μl 0.08% Methocel/H₂O as vehicle andadministered by oral gavage starting from day 3 post immunization (p.i)until termination. Each treatment group consisted of 8 animals: vehiclealone as a negative control, 5 mg/kg body weight DMF twice a day, 15mg/kg body weight DMF twice a day, 15 mg/kg body weight MMF twice a day.The compounds were obtained via Fumapharm AG. Oral gavage was used toensure exact dosing and to avoid compound degradation.

Spinal cord tissues were fixed in 4% paraformaldehyde and embedded inparaffin. Slides were deparaffinized and rehydrated in graded alcoholsolutions. Antigen retrieval was performed by immersing the slides in 10mM Citrate, pH 6.0 for 20 minutes in a pressure cooker at 120 C (Pascal,Dako Cytomation).

Immunohistochemistry was performed using the Dako autostainer asfollows. Endogenous peroxidase was quenched by a 10 minute incubation in3% H₂O₂/Methanol. The rabbit anti Nrf2 antibody C-20 (sc-722, Santa CruzBiotechnology) was added at a 1:250 dilution in Dako Diluent withBackground Reducing Components (Dako #S3022)C-20 antibody was detectedusing the Envision anti rabbit labeled polymer-HRP (Dako #K4003) and DAB(Vector Labs #SK-4100) was used as the chromogenic substrate.Morphometric analysis of Nrf2 immunostaining was performed using ImageJsoftware from NIH.

The results, shown in FIGS. 3 and 4, demonstrate MMF and DMF activationof Nrf2 in vivo.

All publications and patent documents cited herein are incorporated byreference in their entirety. To the extent the material incorporated byreference contradicts or is inconsistent with the present specification,the present specification will supersede any such material.

1.-17. (canceled)
 18. A method of treating multiple sclerosis in a humansubject in need thereof comprising orally administering to the subjectabout 480 mg dimethyl fumarate per day, wherein the dimethyl fumarate isadministered in the form of tablets or capsules, and wherein the tabletsor capsules are provided with a gastric-resistant coating which, oncehaving passed the stomach, is dissolved within a few minutes by thejuice present in the small intestine and releases the dimethyl fumaratefrom the tablets or capsules.
 19. The method of claim 18, wherein thetablets or capsules do not contain a salt of a monoalkyl fumarate. 20.The method of claim 18, wherein the drug for treating multiple sclerosisin said tablets or capsules consists essentially of dimethyl fumarate.21. The method of claim 18 wherein the dimethyl fumarate is administeredin the form of tablets.
 22. The method of claim 19 wherein the dimethylfumarate is administered in the form of tablets.
 23. The method of claim20 wherein the dimethyl fumarate is administered in the form of tablets.24. The method of claim 18 wherein the dimethyl fumarate is administeredin the form of capsules.
 25. The method of claim 19 wherein the dimethylfumarate is administered in the form of capsules.
 26. The method ofclaim 20 wherein the dimethyl fumarate is administered in the form ofcapsules.
 27. The method of claim 18 wherein the dimethyl fumarate isadministered in the form of microtablets in capsules.
 28. The method ofclaim 19 wherein the dimethyl fumarate is administered in the form ofmicrotablets in capsules.
 29. The method of claim 20 wherein thedimethyl fumarate is administered in the form of microtablets incapsules.
 30. The method of claim 18 wherein the 480 mg dimethylfumarate per day is administered in a single dose.
 31. The method ofclaim 19 wherein the 480 mg dimethyl fumarate per day is administered ina single dose.
 32. The method of claim 20 wherein the 480 mg dimethylfumarate per day is administered in a single dose.
 33. The method ofclaim 27 wherein the 480 mg dimethyl fumarate per day is administered ina single dose.
 34. The method of claim 28 wherein the 480 mg dimethylfumarate per day is administered in a single dose.
 35. The method ofclaim 29 wherein the 480 mg dimethyl fumarate per day is administered ina single dose.
 36. The method of claim 18 wherein the 480 mg dimethylfumarate per day is administered in separate administrations.
 37. Themethod of claim 19 wherein the 480 mg dimethyl fumarate per day isadministered in separate administrations.
 38. The method of claim 20wherein the 480 mg dimethyl fumarate per day is administered in separateadministrations.
 39. The method of claim 18 wherein the 480 mg dimethylfumarate per day is administered in separate administrations of equaldoses.
 40. The method of claim 19 wherein the 480 mg dimethyl fumarateper day is administered in separate administrations of equal doses. 41.The method of claim 20 wherein the 480 mg dimethyl fumarate per day isadministered in separate administrations of equal doses.
 42. The methodof claim 27 wherein the 480 mg dimethyl fumarate per day is administeredin separate administrations of equal doses.
 43. The method of claim 28wherein the 480 mg dimethyl fumarate per day is administered in separateadministrations of equal doses.
 44. The method of claim 29 wherein the480 mg dimethyl fumarate per day is administered in separateadministrations of equal doses.
 45. The method of claim 39 wherein the480 mg dimethyl fumarate per day is administered in separateadministrations of two equal doses.
 46. The method of claim 40 whereinthe 480 mg dimethyl fumarate per day is administered in separateadministrations of two equal doses.
 47. The method of claim 41 whereinthe 480 mg dimethyl fumarate per day is administered in separateadministrations of two equal doses.
 48. The method of claim 18 whereinthe multiple sclerosis is relapsing-remitting multiple sclerosis. 49.The method of claim 19 wherein the multiple sclerosis isrelapsing-remitting multiple sclerosis.
 50. The method of claim 20wherein the multiple sclerosis is relapsing-remitting multiplesclerosis.
 51. The method of claim 18 wherein the method furthercomprises administering at least one immunosuppressive orimmunomodulatory compound that does not upregulate the Nrf2 pathway. 52.The method of claim 19 wherein the method further comprisesadministering at least one immunosuppressive or immunomodulatorycompound that does not upregulate the Nrf2 pathway.
 53. The method ofclaim 20 wherein the method further comprises administering at least oneimmunosuppressive or immunomodulatory compound that does not upregulatethe Nrf2 pathway.
 54. The method of claim 18 wherein the tablets orcapsules further comprise another active compound providing additionalor enhanced therapeutic function.
 55. The method of claim 19 wherein thetablets or capsules further comprise another active compound providingadditional or enhanced therapeutic function.
 56. The method of claim 20wherein the tablets or capsules further comprise another active compoundproviding additional or enhanced therapeutic function.
 57. The method ofclaim 21 wherein the tablets are micro-tablets.
 58. The method of claim22 wherein the tablets are micro-tablets.
 59. The method of claim 23wherein the tablets are micro-tablets.
 60. The method of claim 18wherein the 480 mg dimethyl fumarate is not used with other therapeuticagents.
 61. The method of claim 19 wherein the 480 mg dimethyl fumarateis not used with other therapeutic agents.
 62. The method of claim 20wherein the 480 mg dimethyl fumarate is not used with other therapeuticagents.
 63. The method of claim 60 wherein the 480 mg dimethyl fumarateper day is administered in separate administrations of two equal doses,and the multiple sclerosis is relapse-remitting multiple sclerosis. 64.The method of claim 61 wherein the 480 mg dimethyl fumarate per day isadministered in separate administrations of two equal doses, and themultiple sclerosis is relapse-remitting multiple sclerosis.
 65. Themethod of claim 62 wherein the 480 mg dimethyl fumarate per day isadministered in separate administrations of two equal doses, and themultiple sclerosis is relapse-remitting multiple sclerosis.